Abstract: In order to solve the problem of higher nicotine content in flue-cured tobacco leaves produced in some areas, bacterium strain 5-28, which had the potential of degrading nicotine was isolated from tobacco growing soil in Mile, Yunnan using nicotine as the sole source of carbon and nitrogen. The strain 5-28 was identified as Sinorhizobium sp. 5-28 via morphological observation, physio-biochemical property analysis, 16S rRNA sequencing and phylogenetic analysis. The strain 5-28 is tolerant to nicotine at a concentration of 0-2.5 g/L. The optimum condition for nicotine degradation is: nicotine concentration 1.5 g/L, pH 4.0 and 25℃. Almost 80% nicotine would be degraded after cultivation for 24 hours where the initial concentration was 1 g/L, moreover, the resting cells of strain 5-28 would effectively degrade the nicotine in tobacco leaf as well.